Phosphodiesterase and genes thereof

ABSTRACT

The present invention is to provide a novel phosphodiesterase and a gene thereof, specifically, Type 11 phosphodiesterase (PDE11) and a gene thereof, more specifically, a phosphodiesterase selected from (A) a protein having an amino acid sequenced shown by SEQ.ID.NO: 2, SEQ.ID.NO: 4, SEQ.ID.NO: 6 or SEQ.ID.NO: 39, and (B) a protein having an amino acid sequence shown by SEQ.ID.NO: 2, SEQ.ID.NO: 4, SEQ.ID.NO: 6 or SEQ.ID.NO: 39 in which one or several amino acids are deleted, substituted or added, and having an activity of hydrolyzing a cyclic nucleotide, and a gene thereof, and a method of characterizing, identifying and selecting a phosphodiesterase inhibitor by using the same.

TECHNICAL FIELD

The present invention relates to a novel phosphodiesterase and its gene.

BACKGROUND ART

Cyclic nucleotide such as cAMP, cGMP, etc. are involved in regulationsof many in vivo functions as the second messenger in the intracellularsignal transduction (Kukovetz et al., Naunyn Schmiedeberg's Arch.Pharmacol., Vol. 310, pp. 129-138, 1979; Schram et al., Science, Vol.225, pp. 1350-1356, 1984; Ignarro et al., Annu. Rev. Pharmacol.Toxicol., Vol. 25, pp. 171-191, 1985; Martin et al., J. Pharmacol. Exp.,Vol. 237, pp. 539-547, 1986).

Intracellular concentrations of the cAMP and cGMP, changing in responseto an extracellular signal, are regulated by a balance betweenadenylcyclase and guanylcyclase involved in a synthesis thereof, andphosphodiesterase (PDE) involved in a hydrolysis of cyclic nucleotides.

Until recently, many phosphodiesterases have been found from tissues ofmammals which hydrolyze cyclic nucleotides, and they have beenclassified into plural types, according to homology of amino acidsequence, biochemical properties, characterization by an inhibitor, etc.(Beavo, Physiol. Rev., Vol 75, pp. 725-748, 1995).

For example, PDE1 is Ca²⁺/calmodulin dependent PDE and hydrolyses bothcAMP and cGMP. PDE2 is activated by cGMP and hydrolyses both cAMP andcGMP. PDE classified as PDE3 is inhibited by cGMP. PDE4 specificallyrecognizes cAMP as a substrate, and is Rolipram-sensitive. PDE5specifically recognizes cGMP as a substrate. PDE6 is a photoreceptorcGMP-PDE. PDE7 specifically recognizes cAMP as a substrate, and is notsensitive to Rolipram.

Further recently, existences of 3 kinds of novel types of PDE have beenreported. One is called PDE8, specifically recognizing cAMP as asubstrate, and another is called PDE9, specifically recognizing cGMP asa substrate (Soderling et al., Proc. Natl. Acad. Sci. USA, Vol. 95, pp.8991-8996, 1998; Fisher et al., Biochem. Biophys. Res. Commun., Vol.246, pp. 570-577, 1998; Soderling et al., J. Biol. Chem., Vol. 273, pp.15553-15558, 1998; Fisher et al., J. Biol. Chem., Vol. 273, pp.15559-15564, 1998; Hayashi et al., Biochem. Biophys. Res. Commun., Vol.250, pp. 751-756, 1998). These two PDEs are reported to be insensitiveto IBMX (3-isobutyl-1-methylxanthine). Still another one is calledPDE10, recognizing both cAMP and cGMP as a substrate. However, it hasbeen reported to show stronger affinity toward cAMP (Fujishige et al.,J. Biol. Chem., Vol. 274, pp. 18438-18445, 1999; Kotera et al., Biochem.Biophys. Res. Commun., Vol., 261, pp. 551-557, 1999).

Also, PDE is an important target compound for research and developmentin a pharmaceutical field, and research on its inhibitor has beenearnestly carried out. Among the known pharmaceuticals, there have beenfound those having an inhibitory action on PDE, and also, it has beenfound that a specific PDE inhibitor can serve as a useful therapeuticagent.

For example, Milrinone and Zaprinast as a cardiac are inhibitors of PDE3and PDE5, respectively (Harrison et al., Mol. Pharmacol., Vol. 29, pp.506-514, 1986; Gillespie et al., Mol. Pharmacol., Vol. 36, pp. 773-781,1989). Also, Rolipram whose antidepressant activity has been reported isa PDE4 inhibitor (Schneider et al., Eur. J. Pharmacol., Vol. 127, pp.105-115, 1986). PDE4 inhibitor has been also developed and tested as ananti-inflammatory agent or an antasthmatic agent.

On top of that, IBMX is known as a non-selective type inhibitor actingon many types of PDEs. Vinpocetine is known to be a PDE1 inhibitor, EHNA[erythro-9-(2-hydroxy-3-nonyl)adenine] is known to be a PDE2 inhibitor,Dipyridamole is known to be an inhibitor of PDE5 and PDE6. Also,SCH51866((+)-cis-5-methyl-2-[4-(trifluoromethyl)benzyl]-3,4,5,6a,7,8,9,9a-octahydrocyclopent[4,5]imidazo-[2,1-b]purin-4-one;U.S. Pat. No. 5,939,419) is known to be an inhibitor of PDE1 and PDE5,and E4021 (sodium1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl]pyperidine-4-carboxylate;CAS Registration No. 150452-19-0) is known to be an inhibitor of PDE5.

For development of an excellent pharmaceutical with a high therapeuticeffect and less side effect, it is expected to choose an inhibitorhaving a high selectivity toward a certain type of PDE as a target.

Moreover, it has been sought to find a novel type of PDE, being adifferent molecular species from the known ones, for studying a complexmechanism of intracellular signal transduction, and also, for apossibility to become a target molecule of a new therapeutic agent.

An object of the present invention is to provide a novel type ofphosphodiesterase [Type 11 phosphodiesterase (PDE11)] and its gene.Also, it is to provide a novel method for characterizing, identifying orselecting a phosphodiesterase inhibitor. Further, other objects than theabove will be clear from the following descriptions.

DISCLOSURE OF THE INVENTION

The present inventors have isolated from human and rat a full-lengthcDNA which encodes a novel type of phosphodiesterase (also referred toas PDE11 or PDE11A.) which is a different molecular species from theknown ones. Also, they have succeeded in expressing humanphosphodiesterase (also referred to as PDE11 or PDE11A.) in COS cells bya genetic recombination technique and isolating the same. Moreover, theyhave characterized the enzymatic properties, whereby the presentinvention has completed.

That is, the present invention is Type 11 phosphodiesterase (PDE11) andits gene. More specifically, it is phosphodiesterase selected from thefollowing (A) and (B), and a gene or a nucleic acid which encodes saidphosphodiesterase.

(A) a protein having an amino acid sequence shown by SEQ.ID.NO: 2,SEQ.ID.NO: 4, SEQ.ID.NO: 6 or SEQ.ID.NO: 39, and

(B) a protein having an amino acid sequence shown by SEQ.ID.NO: 2,SEQ.ID.NO: 4, SEQ.ID.NO: 6 or SEQ.ID.NO: 39 in which one or severalamino acids are deleted, substituted or added, which has an activity ofhydrolyzing a cyclic nucleotide.

As the gene or the nucleic acid which encodes the phosphodiesterase ofthe present invention, there may be mentioned a gene or a nucleic acidselected from the following (a) and (b):

(a) a gene or a nucleic acid comprising a DNA having a nucleotidesequence shown by SEQ.ID.NO: 1, SEQ.ID.NO: 3, SEQ.ID.NO: 5 or SEQ.ID.NO:38, and

(b) a gene or a nucleic acid comprising a DNA which hybridizes with aDNA having a nucleotide sequence shown by SEQ.ID.NO: 1, SEQ.ID.NO: 3,SEQ.ID.NO: 5 or SEQ.ID.NO: 38 under a stringent condition, and whichencodes a protein having an activity of hydrolyzing a cyclic nucleotide.

Moreover, the present invention is a recombinant vector and a host cellcontaining said gene or said nucleic acid. Moreover, it is a method forcharacterizing, identifying or selecting a phosphodiesterase inhibitorusing the same.

SEQ.ID.NO: 1 of the sequence listing mentioned below represents anucleotide sequence of a cDNA containing an entire coding region of ahuman homologue (human PDE11 gene. Specifically, it is also referred toas a human PDE11A gene.) of the novel PDE gene isolated by the presentinventors, and SEQ.ID.NO: 2 represents an amino acid sequence of thenovel PDE (human PDE11. Specifically, it is also referred to as a humanPDE11A1.) encoded by said full-length cDNA.

SEQ.ID.NO: 3 of the sequence listing mentioned below also represents anucleotide sequence of a cDNA containing an entire coding region of ahuman homologue (human PDE11 gene. Specifically, it is also referred toas a human PDE11A gene.) of the novel PDE gene isolated by the presentinventors, and SEQ.ID.NO: 4 represents an amino acid sequence of thenovel PDE (human PDE11. Specifically, it is also referred to as a humanPDE11A2.) encoded by said full-length cDNA.

SEQ.ID.NOs: 1 and 3 are nucleotide sequences of cDNAs of the two kindsof splicing variants of the human PDE11 gene, and SEQ.ID.NOs: 2 and 4are amino acid sequences of PDE proteins of the each variant.

SEQ.ID.NO: 5 of the sequence listing mentioned below represents anucleotide sequence of a cDNA containing an entire coding region of arat homologue (rat PDE11 gene. Specifically, it is also referred to as arat PDE11A gene.) of the novel PDE gene isolated by the presentinventors, and SEQ.ID.NO: 6 represents an amino acid sequence of thenovel PDE (rat PDE11. Specifically, it is also referred to as a ratPDE11A2.) encoded by said full-length cDNA.

SEQ.ID.NO: 38 of the sequence listing mentioned below also represents anucleotide sequence of a cDNA containing an entire coding region of arat homologue (rat PDE11 gene. Specifically, it is also referred to as arat PDE11A gene.) of the novel PDE gene isolated by the presentinventors, and SEQ.ID.NO: 39 represents an amino acid sequence of thenovel PDE (rat PDE11. Specifically, it is also referred to as a ratPDE11A1.) encoded by said full-length cDNA

SEQ.ID.NOs: 5 and 38 are nucleotide sequences of cDNAs of the two kindsof splicing variants of the rat PDE11 gene, and SEQ.ID.NOs: 6 and 39 areamino acid sequences of PDE proteins of the each variant.

As a result of homology search carried out with respect to thenucleotide sequences shown by the above-mentioned SEQ.ID.NOs: 1, 3, 5and 38, and amino acid sequences shown by the above-mentionedSEQ.ID.NOs: 2, 4, 6 and 39, using known DNA data bases (GenBank andEMBL) and protein data bases (NBRF and SWISS-PROT), there was foundnothing that is expected to be derived from the same kinds of molecularspecies, except for EST (Genbank/EMBL ID No: AI025081).

Moreover, as a result from a comparison between each of the amino acidsequences of the human PDE11 shown by SEQ.ID.NO: 4 and the rat PDE11shown by SEQ.ID.NO: 6, a homology as high as about 93% was confirmed.Also, as a result from a comparison between each of the amino acidsequences of the human PDE11 shown by SEQ.ID.NO: 2 and the rat PDE11shown by SEQ.ID.NO: 39, a homology as high as about 94% was confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of a kinetic analysis on hydrolysis of cAMP orcGMP by human PDE11A1 (Lineweaver-Burk plot).

FIG. 2 shows a result of a kinetic analysis on hydrolysis of cAMP orcGMP by human PDE11A2 (Lineweaver-Burk plot).

FIG. 3 shows a result of studies on an influence of cGMP on cAMPhydrolysis activity, and an influence of cAMP on cGMP hydrolysisactivity, with respect to the human PDE11A1 and the human PDE11A2, wherein relative values (%) of activity are shown, taking a cAMP hydrolysisactivity without adding cGMP, or a cGMP hydrolysis activity withoutadding cAMP as 100%.

FIG. 4 shows kinds of organs and tissues of human for which anexpression of the PDE11 gene was studied by dot blot analysis.

BEST MODE FOR CARRYING OUT THE INVENTION

As the protein of the present invention, there are mentioned thosehaving an amino acid sequence shown by SEQ.ID.NO: 2, 4, 6 or 39. Thereare also mentioned those having an amino acid sequence shown bySEQ.ID.NO: 2, 4, 6 or 39, in which one or several amino acids aredeleted, substituted or added.

Deletion, substitution and addition of the amino acids are admitted aslong as the activity of hydrolyzing cyclic nucleotides is not lost, andnormally, it is from 1 to about 420, preferably, from 1 to about 310,and more preferably, from 1 to about 165, further more preferably, from1 to about 80, and still further preferably, from 1 to about 40.

As a region responsible for the activity of hydrolyzing cyclicnucleotides in PDE11 (PDE11A), that is, as a catalytic region of PDE11(PDE11A), there are exemplified a region corresponding to from the640^(th) to the 881^(st) amino acid residues of the amino acid sequenceshown by SEQ.ID.NO: 2 of the below mentioned sequence listing, a regioncorresponding to from the 390^(th) to the 631^(st) amino acid residuesof the amino acid sequence shown by SEQ.ID.NO: 4, etc.

In order not to loose the activity of PDE11 (PDE11A) for hydrolyzingcyclic nucleotides, it is expected that more amino acid sequences areconserved in a region responsible for the activity of the PDE11(PDE11A), that is, in a catalytic region of the PDE11 (PDE11A) than inother regions.

Deletion, substitution or addition of the amino acids in the catalyticregion of the PDE11 (PDE11A) is, normally from 1 to about 20, preferablyfrom 1 to about 10, more preferably from 1 to about 5. Such a catalyticregion of a protein has a homology with a catalytic region existing inthe amino acid sequence shown in SEQ.ID.NO: 2 or 4, normally by about90% or more, preferably about 95% or more, more preferably about 97% ormore.

On the other hand, deletion, substitution or addition of the amino acidsin a non-catalytic region of PDE11 (PDE11A) is normally from 1 to about400, preferably from 1 to about 300, more preferably from 1 to about160, further preferably from 1 to about 80, yet further preferably from1 to about 40.

These proteins include an artificially modified mutant protein, aprotein derived from other living species, etc., as well as naturallyoccurring mutant proteins.

As the gene or the nucleic acid of the present invention, there arementioned those comprising a DNA having a nucleotide sequence shown bySEQ.ID.NO: 1, 3, 5 or 38. Also, there are mentioned those comprising aDNA which hybridizes with a DNA having a nucleotide sequence shown bySEQ.ID.NO: 1, 3, 5 or 38, under a stringent condition. There is nolimitation for such a hybridizable DNA as long as it encodes a proteinhaving an activity of hydrolyzing cyclic nucleotide. Such a DNA has ahomology with a nucleotide sequence shown by SEQ.ID.NO: 1, 3, 5 or 38,by normally about 70% or more, preferably about 80% or more, morepreferably about 90% or more. Such a gene or a nucleic acid includes anartificially modified mutant gene, a homologous gene derived from adifferent living species, etc., as well as a naturally occurring mutantgene.

In the present invention, hybridization under a stringent conditionmeans carrying out hybridization, in case of a normal stringentconditions, in a hybridization solution of a salt concentration of 6×SSCor an equivalent thereof, at a temperature condition of 50˜70° C. for 16hours, optionally carrying out preliminary washing with a solution of asalt concentration of 6×SSC or an equivalent thereof, and washing in asolution of a salt concentration of 1×SSC or an equivalent thereof.Also, in case of a condition with a higher stringency (highly stringentcondition), the above-mentioned washing is carried out in a solution ofa salt concentration of 0.1×SSC or an equivalent thereof.

The gene or the nucleic acid of the present invention can be isolated byscreening tissues or cells of mammals as a genetic source. As mammals,human as well as non-human animals such as dog, cow, horse, goat, sheep,ape, pig, rabbit, rat and mouse, etc. are mentioned. Among them, it isdesirable to use one of human for a use in research and development of atherapeutic agent for human beings.

The gene or the nucleic acid of the present invention can be obtained byutilizing information on a sequence disclosed in the presentspecification (SEQ.ID.NO: 1, 3, 5 or 38 of the below mentioned sequencelisting). For example, primers and probes are designed based on theinformation on the disclosed nucleotide sequence, and using the same, itcan be chosen and obtained from the DNA library by suitably combiningPCR (polymerase chain reaction) method, colony hybridization method andplaque hybridization method.

For example, cDNA is synthesized from mRNA prepared from cells ortissues of mammals, and using this as a template, cDNA fragment isobtained by PCR method. Using the obtained cDNA as a probe, cDNA libraryis screened by colony hybridization method or plaque hybridizationmethod to obtain a full-length cDNA. Also, genomic DNA can be isolatedby screening genomic DNA library. Further, by screening DNA library ofother mammals, homologous genes from other living species can beisolated.

DNA library such as cDNA library, genomic DNA library, etc. can beprepared according to a method described in, for example, “MolecularCloning” (written by Sambrook, J., Fritsch, E. F. and Maniatis, T.,published by Cold Spring Harbor Laboratory Press in 1989).Alternatively, commercially available libraries can be used if they areavailable.

By determining a nucleotide sequence of the obtained cDNA, a codingregion of the protein as a genetic product can be determined, therebyobtaining an amino acid sequence of this protein.

PDE of the present invention can be produced by overexpression by anusual recombinant DNA technique. Also, it can be produced in a form of afusion protein with other protein or a peptide.

For example, a DNA coding PDE is inserted into a vector so that it islinked downstream of an appropriate promoter, thereby constructing anexpression vector. Subsequently, the obtained expression vector isintroduced in a host cell.

As an expression system (host-vector system), for example, expressionsystems such as bacteria, yeasts, insect cells and mammalian cells canbe mentioned. Among these, for obtaining a functionally well preservedprotein, insect cells (Spodoptera frugiperda SF9, SF21, etc.) andmammalian cells (monkey COS-7 cells, Chinese hamster CHO cells, humanHeLa cells, etc.) are preferably used as a host.

As a vector, in case of the mammalian cell system, retrovirus typevector, papilloma virus vector, vaccinia virus vector, SV40 type vector,etc. can be used, and in case of the insect cell system, baculovirusvector, etc. can be used.

As a promoter, in case of the mammalian cell system, SV40 promoter, LTRpromoter, elongation 1α promoter, etc., and in case of the insect cellsystem, polyhedrin promoter, etc, can be used.

As a DNA coding PDE, a cDNA corresponding to a naturally existing mRNA(for example, those comprising a nucleotide sequence shown by SEQ.ID.NO:1, 3, 5 or 38) can be used, however, it is not limited to this.Alternatively, a DNA corresponding to an amino acid sequence of adesired protein is designed and used. In this case, 1 to 6 kinds areknown for a codon coding each of an amino acid, and codons to be usedmay be chosen randomly. However, for example, by considering a codonusage (frequency) of a host to be used for expression, a sequence with ahigher expression frequency can be designed. A DNA comprising thedesigned nucleotide sequence can be obtained by means of DNA chemicalsynthesis, fragmentation of the above cDNA and linking, partialmodification of the nucleotide sequence, etc. Artificial and partialmodification of the nucleotide sequence or an introduction of a mutationcan be carried out by site specific mutagenesis (Proceedings of NationalAcademy of Sciences, Vol. 81, pp. 5662 to 5666, 1984), etc., using aprimer comprising a synthetic oligonucleotide coding a desiredmodification.

PDE of the present invention can be isolated and purified from acultured product of the cells into which the expression vector isintroduced, optionally combining known purification methods (salting outby inorganic salts, fractional precipitation, ion-exchange resin columnchromatography, affinity column chromatography, gel filtration, etc.).

A nucleic acid (oligonucleotide or polynucleotide) which hybridizes withthe gene or the nucleic acid of the present invention under a stringentcondition can be used as a probe for detecting the gene of the presentinvention. Also, it can be used, for example, as an anti-senseoligonucleotide, a ribozyme, or a decoy for modifying an expression of agene. As such a nucleic acid, for example, a nucleotide comprising apartial sequence of successive 14 bases or more, usually, in thenucleotide sequence shown by SEQ.ID.NO: 1, 3, 5 or 38, or acomplementary sequence thereof can be used.

Using the PDE of the present invention or a protein or peptide having animmunological equivalency thereto (a synthetic peptide containing afragment or a partial sequence of a protein) as an antigen, an antibodywhich recognizes the PDE of the present invention can be obtained.Immunological equivalency means, for example, ability to cross-reactwith an antibody against the PDE of the present invention.

A polyclonal antibody can be prepared by an ordinary method ofinoculating a host animal (for example, rat, rabbit, etc.) with anantigen, and collecting immune serum. A monoclonal antibody can beprepared by an ordinary technique such as a hybridoma method. Further, agene of a monoclonal antibody is modified to prepare a humanizedmonoclonal antibody.

Using the above-obtained antibody, an expression of PDE of the presentinvention in a cell or a tissue can be detected by an ordinaryimmunochemical method (enzyme immunoassay, etc.). Also, by means of anaffinity chromatography using an antibody, purification of PDE of thepresent invention can be carried out.

The fact that PDE of the present invention has an activity ofhydrolyzing a cyclic nucleotide (cAMP or cGMP) can be confirmed by agenerally known method for measuring PDE activity (Thompson et al., Adv.Cyclic Nucleotide Res., Vol. 10, pp. 69-92, 1979; Yanaka et al., Eur. J.Biochem., Vol. 255, pp. 391-399, 1998).

As a substrate for an enzyme reaction, cyclic nucleotide such as cAMP,cGMP, etc. and their derivatives may be used. PDE of the presentinvention recognizes either of cAMP and cGMP as a substrate andhydrolyzes them.

PDE of the present invention can be used for characterization,identification or selection of phosphodiesterases inhibitors.

For example, by carrying out an enzyme reaction in a system containingPDE of the present invention, a substrate for the enzyme and a testsubstance (preferably a compound with a low molecular weight, etc.), aninhibitory action of the test substance on the enzyme activity (anactivity of hydrolyzing a cyclic nucleotide) is determined.

Alternatively, by carrying out a binding reaction in a system containingPDE of the present invention and a test substance (preferably a compoundwith a low molecular weight, etc.), whether or not the test substancehas a binding ability toward PDE of the present invention is determined.A test substance having a binding ability (a ligand) has a highpossibility to serve as an inhibitor.

Moreover, by measuring an inhibitory action (or a binding ability)against PDE of the present invention of the test substance (preferably acompound with a low molecular weight, etc.), and by comparing aninhibitory action (or a binding ability) on other types of PDE,selectivity of the inhibitory action (or the binding ability) can bedetermined. Accordingly, an inhibitor having a relatively high actiontoward a specific type of PDE (a selective inhibitor) can be selected.Also, an identification and characterization of an inhibitor becomepossible.

Hereinafter, the present invention will be explained in more detail byExamples, however, the present invention is not limited to theseExamples.

Incidentally, in Examples below, each operation was carried out, unlessotherwise specified, according to methods described in “MolecularCloning” (written by Sambrook, J., Fritsch, E. F. and Maniatis, T.,published by Cold Spring Harbor Laboratory Press in 1989), or accordingto directions attached to commercially available reagents and kits whenthey were used.

EXAMPLES Example 1 Isolation of cDNA of Human Novel PDE (PDE11) (I)

(1) From a comparison of amino acid sequences of catalytic regions ofvarious known PDE molecules, a highly conserved region was chosen, and asense primer and an anti-sense primer were designed for PCR based on thenucleotide sequence coding this region. As the sense primer, anoligonucleotide was designed comprising a sequence shown by SEQ.ID.NO: 7of the below mentioned sequence listing, and as the anti-sense primer,an oligonucleotide comprising a sequence shown by SEQ.ID.NO: 8 wasdesigned.

RT-PCR (reverse transcript-polymerase chain reaction) was carried outusing these PCR primers to isolate a cDNA fragment from mRNA of humantestis.

That is, a reverse transcript reaction was carried out using mRNA ofhuman testis (available from Clontech), RNA PCR kit (GeneAmp RNA PCRCore kit, available from PE Biosystems) and random primer (hexamer) toobtain cDNA. Using the obtained cDNA as a template, PCR reaction wascarried out using the above-designed oligonucleotides comprisingnucleotide sequences shown in SEQ.ID.NOs: 7 and 8 as a sense primer andan anti-sense primer, respectively. The PCR reaction was repeated for 30cycles in total, one cycle being carried out under conditions of at 94°C. for 30 sec., at 55° C. for 30 sec., and at 72° C. for 30 sec.

The obtained PCR product was linked to a vector plasmid pGEM-T Easy(available from Promega), and a nucleotide sequence thereof wasdetermined. The nucleotide sequence was determined using an automaticDNA sequencer (ABI PRISM 310, available from PE Biosystems), by dideoxymethod (using BigDye terminator cycle sequencing reaction kit availablefrom PE Biosystems) (hereinafter the same as the above). The nucleotidesequence of the thus obtained cDNA fragment was found to be a novelnucleotide sequence which has never been reported, and was found to be anucleotide sequence with a high homology with a part of a cDNA codingPDE5.

(2) From the cDNA fragment obtained in the above (1), full-length cDNAwas obtained by means of RACE method (rapid amplification of cDNA ends)with a procedure described below.

First, based on the information of the nucleotide sequence of cDNAobtained in the above (1), an oligonucleotide comprising a sequenceshown in SEQ.ID.NO: 9 of the below mentioned sequence listing wasdesigned as an anti-sense primer, and using this primer and a kit forRACE for elongation of 5′ end (5′-RACE) (5′-Full RACE Core Set,available from TaKaRa Shuzo), cDNA fragment was prepared from mRNA ofhuman testis (available from Clontech).

Further, using the prepared cDNA fragment as a template, LA PCR (longand accurate PCR) (using LA PCR Kit, available from TaKaRa Shuzo) wascarried out. As the PCR primer, two kinds of oligonucleotides comprisinga sequence shown in SEQ.ID.NOs: 10 and 11 of the below mentionedsequence listing for the first amplification, and for the secondamplification, two kinds of oligonucleotides comprising a sequencesshown in SEQ.ID.Nos: 12 and 13, were used as a sense primer and ananti-sense primer, respectively. Incidentally, each of these 4 kinds ofPCR primers were designed according to the information on the nucleotidesequence of cDNA fragment obtained in the above (1).

A nucleotide sequence of the obtained PCR product was determined.Accordingly, it was found that a part of a missing 5′ end region wasrecovered by the above 5′-RACE, and a nucleotide sequence of therecovered 5′ end region was confirmed.

Next, by further carrying out 5′-RACE (using Marathon-Ready cDNA (humanprostate), available from Clontech) using cDNA derived from humanprostate as a template, a 5′ region was completely recovered. As aprimer, in the first amplification, the first primer (AP1 primer)corresponding to the linker part and a primer comprising a sequenceshown in SEQ.ID.NO: 14 of the below mentioned sequence listing, and inthe second amplification, the second primer corresponding to the linkerpart (AP2 primer) and a primer comprising a sequence shown in SEQ.ID.NO:15 were used, respectively, as a sense primer and an anti-sense primer.These two kinds of anti-sense primers were designed according to theinformation on the nucleotide sequence of the 5′ end region recovered bythe above mentioned 5′-RACE.

Next, 3′-RACE was carried out using a kit for RACE for 3′ end elongation(3′-RACE) (SMART RACE cDNA Amplification kit, available from Clontech)and mRNA of human thyroid gland (available from Clontech) to completelyrecover a 3′ end region. As a PCR primer for the 3′-RACE, in the firstamplification, a primer comprising a sequence shown in SEQ.ID.NO: 16 ofthe below mentioned sequence and the first primer (UPM primer)corresponding to the linker part, and in the second amplification, aprimer comprising a sequence shown in SEQ.ID.NO: 17 and the secondprimer (NUP primer) corresponding to the linker part were used,respectively, as a sense primer and an anti-sense primer. These twokinds of sense primers were designed according to the information on thenucleotide sequence of cDNA obtained in the above (1).

In any of the above RACEs, PCR reaction was repeated for 5 cycles underconditions at 94° C. for 1 min. followed by at 94° C. for 30 sec. and at72° C. for 3 min., 5 cycles under conditions at 94° C. for 30 sec., at70° C. for 30 sec. and at 72° C. for 3 min., and 25 cycles undercondition at 94° C. for 30 sec., at 68° C. for 30 sec. and at 72° C. for3 min.

(3) The nucleotide sequence of the thus obtained full-length cDNA wasanalyzed and compared with the amino acid sequences of the known PDEs toidentify an open reading frame.

Further, based on the information on the nucleotide sequence, PCRprimers were designed enclosing an open reading frame, and RT-PCR wascarried out as described below, using these primers and mRNA of humanprostate.

That is, a reverse transcript reaction was carried out using mRNA ofhuman prostate (available from Clontech), RNA PCR kit (GeneAmp RNA PCRCore kit, available from PE Biosystems) and a random primer (hexamer),to obtain cDNA. Using the thus obtained cDNA as a template, PCR wascarried out. As a PCR primer, an oligonucleotide comprising a nucleotidesequence shown in SEQ.ID.Nos: 18 and 19 of the below mentioned sequencelisting were used as a sense primer and an anti-sense primer,respectively. PCR reaction was done with one cycle being carried outunder conditions of at 94° C. for 30 sec., at 55° C. for 30 sec. and at72° C. for 5 min.

With respect to plural clones obtained from PCR, nucleotide sequences ofcDNA fragments (about 3 kb) were determined, and by comparing each ofthem, errors made by PCR were corrected to confirm a nucleotide sequenceof a full-length cDNA.

(4) The thus obtained full-length cDNA (4476 bp) was thought to be afull-length cDNA of a novel human PDE (referred to as human PDE11 orhuman PDE11A.) gene. The nucleotide sequence was shown in SEQ.ID.NO: 1of the below mentioned sequence listing, and an amino acid sequence of aprotein encoded thereby, that is, human PDE11 (specifically referred toas human PDE11A1.) was shown in SEQ.ID.NO: 2. A molecular weight ofhuman PDE11 (human PDE11A1) estimated from the amino acid sequence (934amino acid residues) was about 105 kDa.

Further, from a homology search with the known PDEs with respect to anamino acid sequence, it was assumed that a catalytic domain of theobtained human PDE11 (human PDE11A1) was a region corresponding to the640^(th) to the 881^(st) amino acid residues, and that a regioncomprising a sequence with a high homology to a cGMP binding regionreported in a literature (McAllister-Lucus, et al., J. Biol. Chem., Vol268, pp 22863-22873, 1993) (hereinafter referred to as cGMP bindingregion) was a region corresponding to the 195^(th) to the 403^(rd) andthe 379^(th) to the 591^(st) amino acid residues, from a homology of thesequence.

When the amino acid sequence of the human PDE11 (human PDE11A1) wascompared to various known human PDEs with cGMP-binding type, homologiesin the catalytic domain were 42% with PDE2A, 51% with PDE5A, 44% withPDE6A, 44% with PDE6B, and 43% with PDE10A. Further, homologies in thetwo cGMP-binding region were, 19 to 47% with PDE2A, PDE5A, PDE6A, PDE6Band PDE10A, respectively.

Example 2 Isolation of cDNA of Human Novel PDE (PDE11) (II)

(1) cDNA was obtained in the same manner as in the above-stated Example1 (1).

(2) RACE was carried out to obtain a full-length cDNA from the cDNAfragment obtained in the above (1), according to the procedure describedbelow.

First, in the same manner as in the above Example 1, a part of the 5′endregion was recovered.

Next, 5′-RACE was further carried out using cDNA derived from humantestis as a template (using Marathon-Ready cDNA (human testis),available from Clontech). As a primer, in the first amplification, thefirst primer (AP1 primer) corresponding to the linker part and a primercomprising a sequence shown in SEQ.ID.NO: 15 of the below mentionedsequence listing, and in the second amplification, the second primercorresponding to the linker part (AP2 primer) and a primer comprising asequence shown in SEQ.ID.NO: 20 were used, respectively, as a senseprimer and an anti-sense primer. These two kinds of anti-sense primerswere designed according to the information on the nucleotide sequence ofthe 5′ end region recovered in the above.

Further, 5′-RACE was carried out again in the same manner as in theabove, to completely recover the 5′ end region. As a primer, in thefirst amplification, AP1 primer and a primer comprising a sequence shownin SEQ.ID.NO: 21 of the below mentioned sequence listing, and in thesecond amplification, AP2 primer and a primer comprising a sequenceshown in SEQ.ID.NO: 22 were used, respectively, as a sense primer and ananti-sense primer. These two kinds of anti-sense primers were designedaccording to the information on the nucleotide sequence of the 5′ endregion recovered by the above mentioned 5′-RACE.

Next, 3′-RACE was carried out in the same manner as in the above Example1, to completely recover a 3′ end region.

(3) After analyzing the nucleotide sequence of the thus obtainedfull-length cDNA, it was compared to an amino acid sequence of the knownPDEs to identify an open reading frame.

Moreover, PCR primers enclosing the open reading frame were designedbased on the information on the nucleotide sequence, and RT-PCR wascarried out using these primers and mRNA of human testis as follows.

That is, a reverse transcript reaction was carried out using mRNA ofhuman testis (available from Clontech), RNA PCR kit (GeneAmp RNA PCRCore kit, available from PE Biosystems) and random primer (hexamer) toobtain cDNA. Using the obtained cDNA as a template, PCR reaction wascarried out. As PCR primers, oligonucleotides comprising nucleotidesequences shown in SEQ.ID.NOs: 23 and 19 were used as a sense primer andan anti-sense primer, respectively. PCR reaction was done with one cyclebeing carried out under conditions of at 95° C. for 30 sec., at 55° C.for 30 sec. and at 72° C. for 3 min.

With respect to plural clones obtained from PCR, nucleotide sequences ofcDNA fragments (about 2.4 kb) were determined, and by comparing each ofthem, errors made by PCR were corrected to confirm a nucleotide sequenceof a full-length cDNA.

(4) The thus obtained full-length cDNA (3507 bp) was thought to be afull-length cDNA of a novel human PDE (referred to as human PDE11 orhuman PDE11A.) gene. The nucleotide sequence was shown in SEQ.ID.NO: 3of the below mentioned sequence listing, and an amino acid sequence of aprotein encoded thereby, that is, human PDE11 (specifically referred toas human PDE11A2.) was shown in SEQ.ID.NO: 4. A molecular weight ofhuman PDE11 (human PDE11A2) estimated from the amino acid sequence (684amino acid residues) was about 78 kDa.

The cDNA of human PDE11 (human PDE11A2) obtained in the present Examplewas assumed to be a splicing variant of the cDNA of human PDE11 (humanPDE11A1) obtained in Example 1. Two kinds of PDEs derived from the twokinds of splicing variants, that is, human PDE11A1 and human PDE11A2were different in N terminals of amino acid sequences (the 1^(st) to the304^(th) amino acid residues in SEQ.ID.NO: 2 and the 1^(st) to the54^(th) amino acid residues in SEQ.ID.NO: 4) and in cDNA sequencescorresponding thereto.

Further, from a homology search with known PDEs with respect to an aminoacid sequence, it was assumed that a catalytic domain of human PDE11A2was a region corresponding to the 390^(th) to the 631^(st) amino acidresidues, and that cGMP binding region of human PDE11A2 was a regioncorresponding to the 129^(th) to the 341^(st) amino acid residues, froma homology of the sequence. The amino acid sequences in the catalyticdomain of human PDE11A2 coincide with those in the catalytic domain ofhuman PDE11A1. Also, the amino acid sequences in the cGMP binding regionof human PDE11A2 coincide with those in one of the two cGMP bindingregions existing downstream (at 3′ side) of the human PDE11A1.

Example 3 Expression and Purification of Human PDE11A1 in COS Cells

(1) Construction of a Vector Plasmid for PDE11A1 Expression

With respect to human PDE11A1 obtained in the above Exmaple 1, cDNAfragment amplified in Example 1 (3) was linked to a vector plasmidpGEM-T Easy (available from Promega) to obtain a plasmid pGEM-PDE11PF.Using this pGEM-PDE11PF as a template, PCR was carried out usingoligonucleotides comprising a nucleotide sequence shown in SEQ.ID.NOs:24 and 25 of the below mentioned sequence listing as a sense primer andan anti-sense primer, respectively. Incidentally, these PCR primers weredesigned based on the 319^(th) to 684^(th) nucleotide sequence ofSEQ.ID.NO: 1 (That is, a nucleotide sequence corresponding to the 1^(st)to the 122^(nd) amino acid residues of SEQ.ID.NO: 2).

cDNA fragment amplified by PCR was linked to pGEM-T Easy (available fromPromega) to obtain a plasmid pGEM-PDE11PBM, and its nucleotide sequencewas confirmed.

Further, cDNA fragments obtained by treating pGEM-PDE11PF withrestriction enzymes KpnI and SalI, and cDNA fragments obtained bytreating pGEM-PDE11PBM with restriction enzymes BamHI and KpnI wereinserted into a BamHI-XhoI site of an expression vector, pcDNA4/HisMaxC(available from Invitrogen: hereinafter referred to as pHis) to obtain avector plasmid pHis-PDE11P for PDE11A1 expression.

(2) Transfection of COS Cells

COS-7 cells (ATCCCRL1651) were subcultured in Dulbecco's modifiedEagle's medium (available from Life Technologies) to which 10% bovinefetal serum, 100 unit/ml penicillin and 100 μM/streptomycin were added,under conditions of at 37° C. and 5% of carbon dioxide.

COS-7 cells were transfected with the above pHis-PDE11 P (or vector pHisfor control) Transfection was carried out using a polycationic liposomeagent (LipofectAMINE PLUS: available from Life Technologies).

(3) Purification of Recombinant Human PDE11A1

After 24 hours from transfection, the cells were washed with an ice coldphosphate buffer, and homogenized by ultrasonic treatment in an ice coldhomogenizing buffer (40 mM Tris-HCl, pH 7.5, 15 mM benzamidine, 5 μg/mlpepstatin A, 5 μg/ml leupeptin). The obtained homogenate was centrifuged(100000 g, 60 min.) to collect supernatant.

The above-obtained supernatant was applied to a nickel-nitrotriacetateresin (available from Qiagen) equilibrated with a buffer solution andincubated at 4° C. for 4 hours. This resin was filled in a column (0.8×5cm) and then, the resin in the column was washed with a washing buffer(40 mM Tris-HCl, pH 7.5, 15 mM benzamidine, 200 mM sodium chloride, 5 mMimidazole, 5 μg/ml pepstatin A, 5 μg/ml leupeptin). Then, proteins wereeluted with an eluting buffer (40 mM Tris-HCl, pH 7.5, 15 mMbenzamidine, 200 mM sodium chloride, 200 mM imidazole, 5 μg/ml pepstatinA, 5 μg/ml leupeptin).

The obtained protein was measured with respect to hydrolysis activity(PDE activity) for cAMP and cGMP, it was found to have a hydrolysisactivity for both cAMP and cGMP.

Incidentally, measurement of PDE activity was done according to aradio-labeled nucleotide method. That is, to 500 μl of a buffer for anassay [50 mM Tris-HCl, pH 8.0, 5 mM magnesium chloride, 4 mM2-mercaptoethanol, 0.33 mg/ml bovine serum albumin (available fromSigma)], containing 1 μM of unlabeled cAMP (or cGMP) and 22 nM of[³H]-cAMP (or [³H]-cGMP) (available from Amersham Pharmacia Biotech),8˜10 μl of the enzyme solution was added to start a reaction. Aftercarrying out a reaction by keeping a temperature at 37° C. for 30minutes, reaction was terminated by boiling the reaction mixture for 2minutes, and 100 μl of 1 mg/ml of snake venom (Crotalus atrox snakevenom) was further added thereto and the temperature was kept at 37° C.for 30 minutes. Subsequently, 500 μl of methanol was added thereto, andthe reaction mixture was applied to Dowex column (1×8-400).Scintillation cocktail was added to each of the eluates, andradioactivity was measured.

Example 4 Expression and Purification of Human PDE11A2 in COS Cells

(1) Construction of a Vector Plasmid for PDE11A2 Expression

With respect to human PDE11A2 obtained in the above Example 2, cDNAfragment amplified in Example 2 (4) was linked to a vector plasmidpGEM-T Easy (available from Promega) to obtain a plasmid pGEM-PDE11TF.Using this pGEM-PDE11TF as a template, PCR was carried out usingoligonucleotides comprising a nucleotide sequence shown in SEQ.ID.NOs:26 and 27 of the below mentioned sequence listing as a sense primer andan anti-sense primer, respectively. Incidentally, these PCR primers weredesigned based on the 100^(th) to 417^(th) nucleotide sequence ofSEQ.ID.NO: 3 (That is, a nucleotide sequence corresponding to the 1^(st)to the 106^(th) amino acid residues of SEQ.ID.NO: 4).

cDNA fragment amplified by PCR was linked to pGEM-T Easy (available fromPromega) to obtain a plasmid pGEM-PDE11TBM, and its nucleotide sequencewas confirmed.

Further, cDNA fragments obtained by treating pGEM-PDE11TF withrestriction enzymes SacI and EcoRV, and EcoRV and SalI, cDNA fragmentsobtained by treating pGEM-PDE11TBM with restriction enzymes BamHI andSacI were inserted into a BamHI-XhoI site of an expression vector, pHis,to obtain a vector plasmid pHis-PDE11T for PDE11A2 expression.

(2) Transfection of COS Cells

COS-7 cells which were subcultured under the same conditions as in theabove Example 3 (2) were transfected with the above-mentionedpHis-PDE11T (or vector pHis for control) in the same manner as in theabove Example 3 (2).

(3) Purification of Recombinant Human PDE11A2

Purification of recombinant human PDE11A2 was carried out in the samemanner as in the above Example 3 (3). Also, when its PDE activity wasmeasured in the same manner as in the above Example 3 (3), it was foundto have a hydrolysis activity for both cAMP and cGMP.

Example 5 Analysis of Enzymatic Properties of Human PDE11

Using each of the purified recombinant human PDE11A1 obtained in Example3 and the purified recombinant human PDE11A2 obtained in Example 4,various enzymatic properties of human PDE11 were analyzed.

(1) Kinetic Analysis of Enzyme Reaction

Enzyme reactions were carried out using a substrate (cAMP or cGMP) ofvarious concentrations, to measure PDE activity (initial reaction rate).Enzyme reaction and PDE activity measurement were done in the samemanner as in the above Example 3 (3), provided that, in a reactionmixture, a concentration of unlabeled cAMP (or cGMP) was set to be 0.1to 10 μM.

The results (Lineweaver-Burk plot) are shown in FIG. 1 for human PDE11A1and FIG. 2 for human PDE11A2.

From the analysis, Km value of human PDE11A1 was 2.96±0.4 5 7 μM, andVmax was 267±47.9 pmol/min/μg of protein, when cAMP was a substrate. Onthe other hand, Km value of human PDE11A1 was 1.43±0.109 μM and Vmax was121±8.08 pmol/min/μg protein, when cGMP was a substrate.

Km value of human PDE11A2 was 2.99±0.488 μM and Vmax was 9.63±1.88pmol/min/μg protein, when cAMP was a substrate. On the other hand, Kmvalue of human PDE11A2 was 1.47±0.115 μM and Vmax was 4.02±0.214 pmol/min/μg of protein, when cGMP was a substrate.

From the above, it was found that although human PDE11 has an activityof hydrolyzing both cAMP and cGMP, it has a higher affinity for cGMP.

(2) Influence of cGMP on cAMP Hydrolysis Activity and Influence of cAMPon cGMP Hydrolysis Activity

It was studied how an addition of cGMP of various concentrations to areaction mixture would affect cAMP hydrolysis activity. It was alsostudied how an addition of cAMP of various concentrations to a reactionmixture would affect cGMP hydrolysis activity.

Enzyme reaction and PDE activity measurement were done in the samemanner as in the above Example 3 (3), except that in an experiment formeasuring cAMP hydrolysis activity, 3.5 μM of unlabeled cAMP was added,and 0.01˜100 μM of cGMP (unlabeled) was added (or was not added). On theother hand, in an experiment for measuring cGMP hydrolysis activity, 1.3μM of unlabeld cGMP was added, and 0.01˜100 μM of cAMP (unlabeled) wasadded (or was not added).

cAMP hydrolysis activity was calculated as a relative value (%) takingan activity measured without adding cGMP as 100%. Also, cGMP hydrolysisactivity was calculated as a relative value (%) taking an activitymeasured without adding cAMP as 100%. The results are shown in FIG. 3.It is clearly confirmed in FIG. 3 that, in case of human PDE11, cAMPhydrolysis activity was lowered depending on a concentration of cGMPadded, while cGMP hydrolysis activity was lowered depending on aconcentration of cAMP added.

(3) Inhibition of Activity by Various Known PDE Inhibitors

Effects of various known PDE inhibitors [IBMX, Vinpocetine, EHNA,Milrinone, Rolipram, Zaprinast, Dipyridamole, SCH51866, and E4021] on acAMP and cGMP hydrolysis activity (PDE activity) of human PDE11 werestudied as follows.

Enzyme reaction was carried out using cAMP or cGMP as a substrate, andvarious kinds of known PDE inhibitors were added in the reaction mixtureto measure a hydrolysis activity. Enzyme reaction and measurement of PDEactivity was done in the same manner as in the above Example 3 (3),except that, in the reaction mixture, in case of adding unlabeled cAMP,a concentration was 3.5 μM, and in case of adding unlabeled cGMP, aconcentration was 1.3 μM, and various kinds of known PDE inhibitors wereadded in an amount of 0˜100 μM.

Inhibitory actions of various PDE inhibitors on an activity of humanPDE11 expressed in terms of IC 50 are shown in Table 1 for human PDE11A1and Table 2 for human PDE11A2, respectively.

TABLE 1 IC50 value (μM) for human PDE11A1 activity Effect on cAMP Effecton cGMP Inhibitor hydrolysis activity hydrolysis activity IBMX 65 ± 1381 ± 16 Vinpocetine >100 >100 EHNA >100 >100 Milrinone >100 >100Rolipram >100 >100 Zaprinast  26 ± 6.8  33 ± 5.3 Dipyridamole 0.82 ±0.28 0.72 ± 0.08 SCH51866  22 ± 1.8  25 ± 5.8 E4021  1.8 ± 0.33  1.8 ±0.25

TABLE 2 IC50 value (μM) for human PDE11A2 activity Effect on cAMP Effecton cGMP Inhibitor hydrolysis activity hydrolysis activity IBMX  30 ± 3.9 38 ± 3.5 Vinpocetine  49 ± 9.2  68 ± 4.0 EHNA >100 >100Milrinone >100 >100 Rolipram >100 >100 Zaprinast  18 ± 1.0  11 ± 3.6Dipyridamole 0.36 ± 0.11 0.34 ± 0.09 SCH51866  11 ± 4.8 8.6 ± 2.7 E40210.88 ± 0.13 0.66 ± 0.19

Example 6 Expressions of PDE11 in Various Tissues of Human

Various kinds of human tissues were examined whether or not PDE11 genewas expressed as follows.

Using mRNAs of various human tissues (Human Multiple Tissue ExpressionArray: available from Clontech), dot blot analysis was done with respectto mRNAs of tissues and organs as shown in FIG. 4. As a probe, a DNAfragment obtained in the above Examples 1 and 2 coding a part of humanPDE11A (a nucleotide sequence of the fragment corresponds to the1237^(th) to the 1801^(st) nucleotide sequence of the human PDE11A1 cDNAshown in SEQ.ID.NO: 1, and also corresponds to the 268^(th) to the832^(nd) nucleotide sequence of the human PDE11A2 cDNA shown inSEQ.ID.NO: 3.) was used by labeling with ³²P. In addition, an amount ofmRNA was controlled to make signal for probes constant, when cDNA ofhuman ubiquitin and major histocompatibility complex class Ic were takenas probes.

Hybridization was carried out under conditions described below. That is,a nylon membrane was placed in a hybridization solution containing aprobe labeled with ³²P (50% formamide, 4×SSC, 0.5% SDS, 5×Denhardt'ssolution, 100 g/ml salmon sperm DNA) at 42° C. for 20 hours forhybridization to proceed. Subsequently, the membrane was washed withwashing solution A (0.5×SSC, 0.1% SDS) at room temperature for 3 min andfurther washed with washing solution B (0.2×SSC, 0.1% SDS) at 60° C. for30 min. for twice. Subsequently, autoradiography was carried out at −80°C. for 3 days.

The result of dot blotting, as shown in FIG. 4, strong expressions ofPDE11 mRNA were detected in prostate and testis. Also, slightly weakexpressions were detected in salivary gland, pituitary gland, thyroidgland and liver (order of intensity of expression was in order of thedescription).

Further, with respect to mRNA of human testis and human prostate,Northern blot analysis was done using the same probe as the above andunder the same conditions.

As a result of Northern blotting analysis, a band of about 3 kb wasdetected in testis, and in prostate, bands of about 2 kb, about 6 kb andabout 10 kb were observed.

Example 7 Isolation of cDNA of Novel Rat PDE (PDE11) (I)

(1) Based on the information on the nucleotide sequence of the cDNA ofhuman PDE11A obtained in the above Examples 1 and 2, PCR primers weredesigned, and RT-PCR was carried out using these to isolate cDNA ofPDE11 from mRNA of rat testis.

That is, reverse transcript reaction was carried out using mRNA of rattestis (available from Wako Junyaku), RNA PCR kit (available from PEBiosystems, GeneAmp RNA PCR Core kit) and random primer (hexamer) toobtain cDNA. Using the obtained cDNA as a-template, PCR was carried out.As PCR primers, oligonucleotides comprising nucleotide sequences shownin SEQ.ID.NOs: 28 and 29 of the below mentioned sequence listing wereused as a sense primer and an anti-sense primer, respectively. The PCRreaction was repeated for 30 cycles in total, one cycle being carriedout under conditions of at 95° C. for 30 sec., at 54° C. for 30 sec.,and at 72° C. for 2 min.

A nucleotide sequence of the cDNA fragment amplified by PCR wasdetermined.

(2) In order to obtain full-length cDNA from the cDNA fragment obtainedin the above (1), 5′-RACE and 3′-RACE were done using mRNA of rat testis(available from Wako Junyaku) according to the below describedprocedures. For RACE, a kit for RACE (available from Clontech, SMARTRACE cDNA Amplification kit) was used.

First, 5′-RACE was done to recover a part of 5′ end region. As primers,in the first amplification, the first primer (UPM primer) correspondingto the linker part and a primer comprising a sequence shown inSEQ.ID.NO: 30 of the below mentioned sequence listing, and in the secondamplification, the second primer corresponding to the linker part (NUPprimer) and a primer comprising a sequence shown in SEQ.ID.NO: 31 wereused, respectively, as a sense primer and an anti-sense primer. Thesetwo kinds of anti-sense primers were designed according to theinformation on the nucleotide sequence of the cDNA obtained in the above(1).

5′-RACE was further carried out to completely recover the 5′ end region.As primers, in the first amplification, the UPM primer and a primercomprising a sequence shown in SEQ.ID.NO: 32 of the below mentionedsequence listing, and in the second amplification, the NUP primer and aprimer comprising a sequence shown in SEQ.ID.NO: 33 were used,respectively, as a sense primer and an anti-sense primer. These twokinds of anti-sense primers were designed according to the informationon the nucleotide sequence of the above-recovered 5′ end region.

Next, 3′-RACE was done to completely recover a 3′ end region. Asprimers, in the first amplification, a primer comprising a sequenceshown in SEQ.ID.NO: 34 of the below mentioned sequence listing and theUPM primer, and in the second amplification, a primer comprising asequence shown in SEQ.ID.NO: 35 and the NUP primer were used,respectively, as a sense primer and an anti-sense primer. These twokinds of anti-sense primers were designed according to the informationon the nucleotide sequence of the cDNA fragment obtained in the above(1).

(3) After analyzing the nucleotide sequence of the thus obtainedfull-length cDNA, it was compared to an amino acid sequence of the knownPDE to identify an open reading frame.

Further, PCR primers enclosing the open reading frame were designedbased on the information on the nucleotide sequence, and RT-PCR wascarried out using these primers and mRNA of rat testis as follows.

That is, a reverse transcription reaction was carried out using mRNA ofrat testis (available from Wako Junyaku), RNA PCR kit (GeneAmp RNA PCRCore kit, available from PE Biosystems) and a polydT-tailed primer toobtain cDNA. Using the obtained cDNA as a template, PCR reaction wascarried out. As a PCR primer, oligonucleotides comprising nucleotidesequences shown in SEQ.ID.NOs: 36 and 37 were used as a sense primer andan anti-sense primer, respectively. PCR reaction was repeated for 30cycles in total, one cycle being carried out under conditions of at 95°C. for 30 sec., at 57° C. for 30 sec. and at 72° C. for 3 min.

With respect to plural clones obtained from PCR, nucleotide sequences ofcDNA fragments (about 2.3 kb) were determined, and by comparing each ofthem, errors made by PCR were corrected to confirm a nucleotide sequenceof a full-length cDNA.

(4) The thus obtained full-length cDNA (3492 bp) was thought to be afull-length cDNA of a novel rat PDE (referred to as rat PDE11 or ratPDE11A.) gene. The nucleotide sequence was shown in SEQ.ID.NO: 5 of thebelow mentioned sequence listing, and an amino acid sequence of aprotein encoded thereby, that is, rat PDE11 (specifically referred to asrat PDE11A2.) was shown in SEQ.ID.NO: 6. A molecular weight of rat PDE11(rat PDE11A2) estimated from the amino acid sequence (685 amino acidresidues) was about 78 kDa.

When the amino acid sequence of rat PDE11 (rat PDE11A2) was compared tothe amino acid sequence of human PDE11A2 obtained in Example 2, ahomology as high as about 93% was confirmed.

Example 8 Isolation of cDNA of Novel Rat PDE (PDE11) (II)

(1) 5′-RACE was carried out using cDNA derived from rat liver (availablefrom Clontech, Marathon-Ready cDNA (rat liver) was used). As primers, inthe first amplification, the first primer (AP1 primer) corresponding tothe linker part and a primer comprising a sequence shown in SEQ.ID.NO:32 of the below mentioned sequence listing, and in the secondamplification, the second primer corresponding to the linker part (AP2primer) and a primer comprising a sequence shown in SEQ.ID.NO: 33 wereused, respectively, as a sense primer and an anti-sense primer. Thesetwo kinds of anti-sense primers were designed according to theinformation on the nucleotide sequence of the cDNA of the rat PDE11 (ratPDE11A2) obtained in the above Example 7.

5′-RACE was further carried out using the cDNA of rat liver (availablefrom Clontech, Marathon-Ready cDNA (rat liver) was used) to completelyrecover the 5′ end region. As primers, in the first amplification, theAP1 primer and a primer comprising a sequence shown in SEQ.ID.NO: 40 ofthe below mentioned sequence listing, and in the second amplification,the AP2 primer and a primer comprising a sequence shown in SEQ.ID.NO: 41were used, respectively, as a sense primer and an anti-sense primer.These two kinds of anti-sense primers were designed according to theinformation on the nucleotide sequence of the 5′ end region recovered inthe above 5′-RACE.

(2) After analyzing the nucleotide sequence of the thus obtainedfull-length cDNA, it was compared to an amino acid sequence of the knownPDE to identify an open reading frame.

Further, PCR primers enclosing the open reading frame were designedbased on the information on the nucleotide sequence, and RT-PCR wascarried out using these primers and mRNA derived from rat liver asfollows.

That is, a reverse transcript reaction was carried out using mRNA of ratliver (available from Clontech), RNA PCR kit (GeneAmp RNA PCR Core kit,available from PE Biosystems) and random primer (hexamer) to obtaincDNA. Using the obtained cDNA as a template, PCR reaction was carriedout. As PCR primers, oligonucleotides comprising nucleotide sequencesshown in SEQ.ID.NOs: 42 and 37 were used as a sense primer and ananti-sense primer, respectively. PCR reaction was repeated for 30 cyclesin total, one cycle being carried out under conditions of at 95° C. for30 sec., at 57° C. for 30 sec. and at 72° C. for 4 min.

With respect to plural clones obtained from PCR, nucleotide sequences ofcDNA fragments (about 3.0 kb) were determined, and by comparing each ofthem, errors made by PCR were corrected to determine a nucleotidesequence of a full-length cDNA.

(3) The thus obtained full-length cDNA (4170 bp) was thought to be afull-length cDNA of a novel rat PDE (rat PDE11A) gene. The nucleotidesequence was shown in SEQ.ID.NO: 38 of the below mentioned sequencelisting, and an amino acid sequence of a protein encoded thereby, thatis, rat PDE11 (specifically referred to as rat PDE11A1.) was shown inSEQ.ID.NO: 39. A molecular weight of rat PDE11 (rat PDE11A1) estimatedfrom the amino acid sequence (935 amino acid residues) was about 105kDa.

When the amino acid sequence of rat PDE11 (rat PDE11A1) was compared tothe amino acid sequence of human PDE11A1 obtained in Example 1, ahomology of as high as about 94% was confirmed.

INDUSTRIAL APPLICABILITY

The novel PDE of the present invention and the gene thereof are usefulfor study of a complex mechanism of intracellular signal transduction.Also, it can possibly serve as a target compound of a therapeutic agentfor novel disease.

Additionally, a method for characterizing, identifying and selecting aninhibitor, using the novel PDE of the present invention and the genethereof is useful for development of an inhibitor with a highselectivity and an excellent pharmaceutical with a high therapeuticeffect and less side effect.

1. An isolated protein having the amino acid sequence shown by SEQ IDNO:2.
 2. The protein according to claim 1, wherein said protein is ahuman protein.
 3. The protein according to claim 1, wherein said proteinis a recombinant protein.